1. Field of the Invention
This invention relates to an apparatus and method for monitoring cells which have been cultured onto electrodes of a microfabricated array. More particularly, it relates to an apparatus and method for monitoring various characteristics of such cells, such as cellular health, cellular adhesion to an electrode or substrate, and changes in cellular impedance, action potential parameters, membrane capacitance, membrane conductance, and cell/substrate seal resistance. It also relates to a technique for evaluating the effect of an analyte (which analyte can include pharmaceutical agents and toxins) upon a cell, and a technique for quality control testing of wet electrochemical systems which utilize microelectrodes.
2. Description of Related Art
Biosensors--sensors including biological materials employed to detect and/or monitor an environment--offer several advantages. Biosensor development can utilize the highly sensitive nature of biological materials to detect directly the presence or absence of analytes by their affect upon cellular metabolism. Thus, for example, utilizing cultured cell systems it is possible to screen for a broad range of toxins, achieving a fast response time, while maintaining high sensitivity. Moreover, a degree of selectively may be achieved by choice of cell type.
Cellular metabolism refers to the orchestration of the chemical and enzymatic reactions that constitute the life process of a cell. These reactions include a vast number of different chemical and enzymatic reactions, relating to the growth and maintenance of the cell. Contemplation of the wide variety of functions that a cell carries out--DNA replication, DNA transcription, RNA translation, digestion of macromolecules, construction of macromolecules, and monitoring of the extracellular and intracellular milieus--provides one with a profound appreciation of the complexity of cellular metabolism. These chemical and enzymatic reactions occur simultaneously within an active cell. Moreover, these reactions do not take place in isolation; rather the pace of each reaction is regulated, in turn, by the product of one or more other reactions. Overall, the organization of cellular metabolism is embedded in a vast network of inter-related cellular reactions. Given this interdependency, it is apparent that analytes that affect one or more aspects of cellular metabolism are likely to manifest their impact on characteristics of the cell, including impedance; action potential parameters, including action potential rate, action potential amplitude, and action potential shape, among others; membrane conductance, and membrane capacitance.
Because of the above advantages, biosensors including live, intact cells (referred to as hybrid biosensors) have several commercially significant applications. For example, such biosensors are particularly useful in detecting chemical and biological warfare (CBW) agents. Biosensors may also supplement existing methods for pharmaceutical screening. It is possible that this type of biosensor technology will eliminate, or at least greatly reduce, animal testing employed in pharmaceutical screening.
Some progress in the development of hybrid biosensor technology has been described within the scientific literature. For example, techniques have been described by Giaever and Keese (in conjunction with others) to monitor the impedance characteristics at cell/electrode interfaces. See M. Kowolenko et al., "Measurement of Macrophage Adherence and Spreading with Weak Electric Fields," Journal of Immunological Methods, 127, 71-77 (1990); C. R. Keese et al., "A Whole Cell Biosensor Based on Cell-Substrate Interactions," Annual International Conference of the IEEE Engineering in Medicine and Biology Society, Vol. 12, No. 2, (1990); P. M. Ghosh et al., "Monitoring Electropermeabilization in the Plasma Membrane of Adherent Mammalian Cells," Biophys. Journal, 64, 1602-09 (1993); C. R. Keese et al., "A Biosensor that Monitors Cell Morphology with Electrical Fields," IEEE Engineering in Medicine and Biology, June/July 1994, 402-08; I. Giaever et al., "Use of Electric Fields to Monitor the Dynamical Aspect of Cell Behavior in Tissue Culture," IEEE Transactions on Biomedical Engineering, Vol. BME-33, No. 2, Feb. 1986; U.S. Pat. No. 4,054,646; U.S. Pat. No. 4,920,047; and U.S. Pat. No. 5,187,096. However, the systems described by Giaever and Keese utilize large area (e.g., 250 .mu.m diameter) electrodes which cannot be completely covered by a single cell. Therefore, accurate measurement of an individual cell's impedance characteristics is interfered with by the parallel impedance of the uncovered electrode area and the impedance of cell-cell contact areas (due to the space between adjacent cells). Moreover, the efforts of Giaever and Keese reveal only motility changes in cells. Further, cellular membranes are modeled as having a constant capacitance and conductance; any changes in capacitance and conductance are explained in terms of varying cellular membrane area.
In Lind et al., "Single Cell Mobility and Adhesion Monitoring Using Extracellular Electrodes" Biosensors and Bioelectronics, 6, 359-67 (1991), a system using relatively large area electrodes similar to that described above was employed, as well as a system utilizing electrodes smaller than the cell to be monitored. In this system, single cell effects could be examined without the shunting effects described above, but this system was only used to monitor cell motility by means of changes in the impedance. Moreover, in such systems membrane conductance and capacitance was presumed to be constant.
Accordingly, there remains a need for a technique that utilizes electrodes smaller than a cell's diameter, but which provides for monitoring changes in cellular membrane capacitance and conductance. Such a technique would also permit monitoring of activation of voltage-gated ionic channel conductance. In addition, such a technique would permit detection and monitoring of compounds that affect the impedance, action potential parameters, membrane conductance, membrane capacitance of a cell, and cell/substrate seal resistance.
Voltage-gated Na.sup.+ channels (among other ion channels) help make nerve cells electrically excitable and enable them to conduct action potentials. When the membrane of a cell with many Na.sup.+ channels is partially depolarized by a momentary stimulus, some of the channels promptly open, allowing Na.sup.+ ions to enter the cell. The influx of positive charge depolarizes the membrane further, thereby opening more channels, which admit more Na.sup.+, causing still further depolarization. This process continues in a self-amplifying fashion until the membrane potential has shifted from its resting value of about -70 mV all the way to the Na.sup.+ equilibrium potential of about +50 mV. At that point, where the net electrochemical driving force for the flow of Na.sup.+ is zero, the cell would come to a new resting state with all its Na.sup.+ channels permanently open, if the open channel conformation were stable. The cell is saved from such a permanent electrical spasm by the automatic inactivation of the Na.sup.+ channels, which now gradually close and stay closed until the membrane potential has returned to its initial negative resting value. The whole cycle, from initial stimulus to return to the original resting state, takes a few milliseconds or less.
In many types of neurons, though not all, the recovery is hastened by the presence of voltage-gated K.sup.+ channels in the plasma membrane. Like the Na.sup.+ channels, these channels open in response to membrane depolarization, but they do so relatively slowly. By increasing the permeability of the membrane to K.sup.+ just as the Na.sup.+ channels are closing through inactivation, the K.sup.+ channels help to bring the membrane rapidly back toward the K.sup.+ equilibrium potential, so returning it to the resting state. The repolarization of the membrane causes the K.sup.+ channels to close again and allows the Na.sup.+ channels to recover from their inactivation. In this way the cell membrane can be made ready in less than a millisecond to respond to a second depolarizing stimulus.
Examining the membrane potential relative to time, an action potential exhibits various characteristics or parameters, including action potential rate (if cells spontaneously depolarize), action potential amplitude, and action potential shape, among others. Action potential rate refers to the frequency with which a cell produces an action potential (rapid depolarization). Action potential amplitude refers to the height of the peak depolarization that occurs in the course of the action potential. Action potential shape refers to the time course of the depolarization and repolarization.
There is also a need in the art for an apparatus for monitoring cells employing an electrode array having reduced electrode diameter and low electrode impedance, thus allowing for detection and monitoring of changes in the cellular membrane of an individual cell.
These results, among others, had not been achieved in the prior art.